Transformations of a Large Aggregate of Hydroxycinnamate Hydroxylase to Lower Molecular Weight Forms by Sulfhydryl Agents in Green Leaves of Sorghum

Abstract

The large, partly microsomal aggregate containing 4-hydroxycinnamate hydroxylase activity isolated from green leaves of Sorghum bicolor at pH 6 was obtained instead as intermediate molecular weight forms when green leaves were ground in the presence of 10 mm mercaptoethanol or dithiothreitol. Elution profiles from agarose (Bio-Gel A-15m and A-1.5m) columns indicated that the 4-hydroxycinnamate hydroxylase activity was due either to multiple forms or to a mixture of forms in various stages of dissociation, the largest being eluted just after the void volume from an agarose 1.5m column. The larger form was similar to the major one found previously in etiolated leaves and was precipitated in the same ammonium sulfate fraction. The activity was unstable, but could be reactivated by incubation of the undiluted enzyme extract alone at 30 C prior to the assay. The data indicate that disulfide bonds are involved in the in vivo formation of the large aggregate in green leaves as well as being necessary for the maintenance of optimal activity of smaller polymeric forms in vitro.